Methods for quantitative determination of heteronuclear compounds using nuclear magnetic resonance spectroscopy, reference materials, and methods for determining the degree of deuteration of deuterated compounds.

Abstract

This invention relates to a method for quantitatively determining heteronuclear compounds using nuclear magnetic resonance spectroscopy, its reference materials, and a method for determining the deuteration degree of deuterated compounds. The invention discloses a method for determining the deuteration degree of a first deuterated compound, the method comprising the steps of: preparing a mixture of the first deuterated compound and a second deuterated compound having a known deuteration degree, such that both deuterated compounds are dissolved; generating a 1H NMR spectrum from the mixture and determining the integral of at least one peak of each of the two deuterated compounds; generating a 2H NMR spectrum from the mixture and determining the integral of the same peak; and calculating the deuteration degree of the first deuterated compound using the determined integral value and the known deuteration degree of the second deuterated compound. The method of this invention makes it possible to perform quantitative NMR spectroscopy without adding the specific standard materials that have been used until now.

Description

[0001] This application is a divisional application of international application PCT / EP2019 / 055520, which entered the Chinese national phase on September 7, 2020, with application number 201980017595.2 and invention title "Method for quantitative determination of heteronuclear compounds by nuclear magnetic resonance spectroscopy, its reference material and method for determining the degree of deuteration of deuterated compounds". Technical Field

[0002] This invention relates to a method for quantitatively determining an analyte in a test sample using an NMR spectrometer, wherein a deuterated solvent used to dissolve the analyte is used as an internal standard, a reference material for the method of the invention, and a method for determining the degree of deuteration of a deuterated compound. Background Technology

[0003] Nuclear magnetic resonance spectroscopy, also referred to below as NMR spectroscopy, is one of the fundamental methods for determining the structure of organic compounds. It is based on the fact that the nuclei of many elements have non-zero nuclear spin, and that the angular momentum gained when an external magnetic field is applied allows for descriptions of the chemical environment of those atoms.

[0004] Atoms with zero nuclear spin (I = 0), for example 12 C or 16 O cannot be detected by NMR spectroscopy. Conversely, all atoms with non-zero nuclear spin can be detected by NMR spectroscopy. A nuclear spin of 1 / 2 is most favorable because only two possible eigenstates exist in this case, m = +1 / 2 and m = -1 / 2. The number of possible states is calculated using the formula: 2I + 1. Therefore, when using NMR spectroscopy in organic chemistry, the most suitable nucleus is... 1 H, 13 C 15 N、 19 F, 29 Si、 31 P and 77 Se.

[0005] Beyond pure structural analysis, NMR spectroscopy can also be used for the quantitative determination of compounds under investigation, i.e., analytes. In quantitative NMR spectroscopy, the concentration of the analyte in the sample solution is calculated by comparing the signal intensities of the analyte and a simultaneously measured reference material. Typically, an internal standard is added to the sample solution as a reference material; the purity of the internal standard is known, and its signal should not overlap with those of the analyte. The reference material can also be used as an external standard. In the following text, the reference material used in NMR spectroscopy is also referred to as a "standard".

[0006] In quantitative assessment using NMR spectroscopy, the signals of the analyte and the reference material are integrated separately. It is assumed that each atom exhibiting a signal in the spectrum contributes an equal component to the integral of the signal, thus the number of atoms is proportional to the integral area. The mass of the analyte in the sample solution can be calculated from the integration ratio, taking into account the mass and molar mass of the analyte and the reference material, as well as the number of atoms whose signals are integrated.

[0007] Unlike other chromatographic methods, quantitative NMR spectroscopy does not require that the reference material used as a standard be identical to the analyte. The analyte and standard only need to contain the same type of atoms (the same nuclides), whose signals are recorded in the NMR experiment. This makes it possible to use internal standards as described above, wherein the internal standard is optimally selected so that its signal does not overlap with the analyte signal in the NMR spectrum. A predetermined amount of the analyte and standard (determined by measurement or weighing) is dissolved in a sufficient volume of a commonly used deuterated solution, and then measured in an NMR spectrometer. For this method, it is not necessary to know the exact amount of solvent used.

[0008] For each nucleus to be examined, appropriate standards (standard substances) are stored to varying degrees as precisely defined standard solutions. If other nuclide types of the same sample are to be determined, additional standards must be added, which is very time-consuming because the standard solutions must be prepared and precisely measured. Furthermore, the purity of these additional standard solutions must be checked periodically, which also means extra effort.

[0009] To reduce the number and workload of different standards and to provide a universal standard for most important NMR active nuclides of organic molecules, a multinuclear standard was proposed in DE102012204701A1. This multinuclear standard contains phosphorus, nitrogen, and fluorine atoms in addition to carbon and hydrogen atoms. By using a single standard, several types of nuclides can be measured on the same sample without further intervention. Another advantage of multinuclear standards is that all types of atoms contained therein are always present in accurate stoichiometric ratios, thus eliminating weighing errors when determining the atomic ratios in the standard.

[0010] However, if it is necessary to measure other reactive nuclides, especially those from the range of inorganic anions and cations, such as sodium (Na), potassium (K), chlorine (Cl), bromine (Br), or aluminum (Al), no suitable substance can be used as a standard. Na exists only as a simple cation, as do K or Ca. Iodine (I), Br, and Cl are only stable in their anionic form in water and organic solutions. In principle, only Cl can be used as an internal standard in the form of perchlorate. Conversely, periodate reacts too much in the presence of iodides to be detectable and therefore can only be used as an external standard, which has corresponding drawbacks. In such cases, calibration lines must be established, leading to many cumbersome reference measurements. Another fundamental problem with external calibration in NMR spectroscopy is the lack of a quantifiable measuring element. For example, in HPLC chromatography, such an element is used for UV detection, where a permanently usable measuring chamber or a replaceable standardized analytical cup is used. Commercially available NMR measuring tubes are not standardized in volume and have an error of 0.5% to 10% based on mass. Alternatively, the same test tube could be used for each measurement, but this results in time-consuming rinsing and quality checks, and is impractical, especially for serial and routine measurements.

[0011] In all past attempts to overcome these drawbacks, each quantitative determination required at least a second measurement, resulting in high uncertainty. The ERETIC (Electron Reference for In Vivo Concentration) procedure attempted to quantify using a radiation electron reference signal. However, this work failed due to volume variations. Even with the PULCON (Pulse Length-Based Concentration Determination) method, results could not be obtained more precise than allowed by the standard deviation of the average volume of the NMR tubes. A second problem is that each NMR tube must be individually tuned according to sensitivity, a process known as “tuning and matching.” Volume variations and matching introduce large measurement uncertainties. Summary of the Invention

[0012] Therefore, the object of this invention is to overcome the shortcomings of the aforementioned quantitative NMR spectroscopy methods and provide a nuclear magnetic resonance spectroscopy method, i.e., NMR spectroscopy, which allows for the simple yet accurate quantitative determination of all NMR-active nuclides. Another object of this invention is to provide the necessary identifying reference material for the NMR spectroscopy method.

[0013] This task is solved by the method described in this invention, which uses an NMR spectrometer to quantitatively determine the analytes in the sample. The method of this invention includes the following steps:

[0014] A sample solution is provided by dissolving a defined amount of the test sample in a defined amount of deuterated solvent.

[0015] The NMR spectrometer is calibrated by determining calibration factors associated with a first NMR active nuclide and a second NMR active nuclide, wherein the first NMR active nuclide is a component of the deuterated solution and the second NMR active nuclide is a component of the analyte.

[0016] The first NMR spectrum was generated by performing a first NMR experiment on the sample solution, and the resonance frequency of the first NMR active nuclide was determined.

[0017] A second NMR spectrum was generated by performing a second NMR experiment on the sample solution, and the resonance frequencies of the active nuclides in the second NMR spectrum were determined.

[0018] Taking into account the signals in the first and second spectra and the calibration factor, calculate the mass of the analyte in the sample solution.

[0019] The first and second NMR active nuclides are different from each other.

[0020] The calibration factor is determined as follows: the resonance frequencies of the first and second NMR active nuclides are measured in a calibration solution containing known amounts of the first and second NMR active nuclides, generating two calibration spectra; and the signals of the first and second NMR active nuclides in the calibration spectra are compared.

[0021] The first and second NMR experiments, as well as the determination of the calibration factor, were performed using the same NMR spectrometer.

[0022] This invention is based on the inventors' discovery that, in quantitative NMR spectroscopy, the deuterated solvent used to prepare the sample solution can also be used as an internal standard, thereby enabling the quantitative determination of all NMR-active nuclides in the sample solution. Therefore, the method of this invention can also be used for the quantitative detection of nuclides that are not components of the standard used. Hereinafter, this method is referred to as "heteronuclear quantitative determination" or "heteronuclear quantification".

[0023] Therefore, the method of the present invention is characterized in that the first NMR active nuclide and the second NMR active nuclide are different from each other. The designation "first" or "second" NMR active nuclide is used only to distinguish between two different NMR active nuclides.

[0024] Therefore, this invention differs from conventional quantitative NMR spectroscopy, in which the same nuclide is quantitatively detected in both the standard and the analyte. Thus, for distinction, commonly used quantitative NMR spectroscopy can be described as "isonuclear quantitative determination" or "isonuclear quantification."

[0025] The NMR active nuclides referred to in this application are all nuclides that have non-zero nuclear spin and are therefore detectable by NMR spectroscopy, namely, the atomic nuclei of a single nuclide or different isotopes of an element.

[0026] The method of the present invention is used to determine the proportion of a specific substance (analyte) in a mixture (test sample) of several substances.

[0027] In order to make a quantitative description using the method of the present invention, when preparing the sample solution to be measured, a certain amount of the test sample must be dissolved in a certain amount of deuterated solvent. The certain amounts of the test sample and the deuterated solvent can be obtained independently by measuring the required volume or weighing the required mass. Therefore, the mass of the test sample and the deuterated solvent contained in the sample solution is known.

[0028] The statement that the test sample is dissolved in the deuterated solvent means that the test sample is preferably completely dissolved in the solvent. However, it is sufficient if only the analytes contained in the test sample are completely dissolved.

[0029] Preferably, the sample solution is provided in a container suitable for NMR spectroscopy, such as an NMR test tube.

[0030] D₂O, DMSO-d₆, CDCl₃, acetone-d₆, acetonitrile-d₃, and benzene-d₆ are preferably used as deuteration solvents, but in principle all deuteration solvents can be used. The deuteration solvent used is appropriately selected based on different parameters, such as the solubility of the test sample or analyte in the solvent or the inertness of the solvent.

[0031] In the method of this invention, the prepared sample solution is subjected to two NMR experiments, wherein the resonance frequencies of the first and second NMR active nuclides are determined and the results are presented in the corresponding NMR spectra. In principle, the order of the first and second NMR experiments is irrelevant. However, the two NMR experiments must be performed on the same sample solution. This means that the ratio of deuterated solvent to analyte in the sample solution must remain unchanged between the two NMR experiments. Only in this way can a quantitative description of the amount of analyte in the sample be made.

[0032] The statement that an NMR active nuclide is a "component of the deuterated solvent" means that the nuclide is represented in the chemical formula (total molecular formula) of the solvent. Similarly, the statement that an NMR active nuclide is a "component of the analyte" means that the nuclide is represented in the chemical formula (total molecular formula) of the analyte. For example, if D₂O is used as the deuterated solvent, then deuterium ( 2 H) is a component of the deuterated solvent. If the analyte is, for example, ethanol (C2H5OH), then both carbon (C) and hydrogen (H) are components of the analyte.

[0033] However, this does not preclude the presence of one or both of these nuclides in both total chemical formulas. For example, if the method of the present invention is used for the quantitative determination of organic hydrocarbon compounds using acetone-d6 as a deuterating solvent, carbon (C) is both a component of the deuterating solvent and a component of the analyte.

[0034] Two NMR experiments are performed in the usual manner, and corresponding NMR spectra are generated from the results. One spectrum shows the signal of the NMR-active nuclide in the deuterated solvent used as an internal standard, and the other spectrum shows the signal of the NMR-active nuclide in the analyte. For example, NMR spectra can be generated / prepared in the first NMR experiment. 2 H NMR spectra, and can be generated / prepared in the second NMR experiment. 1 H NMR spectrum or 13 C NMR spectroscopy.

[0035] In contrast, in typical homonuclear quantitative assays, only a single NMR experiment is performed and only a single NMR spectrum is obtained, which contains both the signal of the internal standard and the signal of the analyte.

[0036] Therefore, in the method of the present invention, measurements from two different NMR experiments are used to quantitatively determine the analyte in the test sample. The difference between the two NMR experiments lies in that the resonances of different nuclides were measured at different resonance frequencies. For quantitative purposes, the measurement results obtained by this method, i.e., signal integrals from different spectra, cannot be directly compared, as explained below.

[0037] A phenomenon in quantum physics is that, under the same magnetic field strength, different nuclei exhibit different sensitivities due to their different resonance frequencies. This effect is quantified by the gyromagnetic ratio γ of the nuclide, detected by NMR spectroscopy, and expressed as a sensitivity value. These values ​​are usually known. For example, 1 H is the most sensitive nuclide. The next most sensitive nuclide is... 19 F, its sensitivity is already higher than 1 H decreased by 17%. 2 H's sensitivity compared to 1 H is now only 1% (9.65 × 10⁻⁶) -3 ).

[0038] When the NMR-active nuclide under study is not the only naturally occurring isotope of the element, the relative frequency of the nuclide in a naturally occurring mixture of isotopes also plays a role. This factor is considered in sensitivity, which is the product of sensitivity and the natural frequency of the nuclide. For example, nuclei 31 P, 19 F and 23Na is 100% monoisotopic. Under conditions of no significant error, 1 H can be similarly considered as a monoisotope. On the other hand, for example, the nucleus... 15 N and 13 The natural frequencies of C must be taken into account. These values ​​are usually known.

[0039] In the method of the present invention, the deuterated solvent used as an internal standard is typically highly enriched in terms of deuterium content, with a deuteration degree between 99.5% and 99.99%, i.e., almost 100%. Alternatively, using the method of the present invention, by comparison... 1 H signal and 2 The H signal (and by knowing the corresponding calibration factor) allows for a simple, rapid, and reliable determination of the degree of deuteration of the solvent used.

[0040] If generated in the first NMR experiment 2 H NMR spectroscopy, and at a deuteration level of approximately 100%, relative to 1 NMR signal measurement of H, 2 The sensitivity for measuring the NMR signal of H is approximately 9.65 × 10⁻⁶. -3 .

[0041] Beyond the aforementioned quantum mechanical and chemical factors, instrument-specific factors also make it difficult to directly compare measurements from different NMR experiments. These factors arise from the geometry of the measurement apparatus and are individual and specific to each NMR spectrometer, even when comparing different instruments of the same design. Therefore, the spectrometer's coil geometry and other hardware parameters have been shown to have identifiable effects. Consequently, instrument-specific factors for each NMR spectrometer must be determined empirically, which is not particularly complex. They remain constant as long as the measurement apparatus is not altered. The determination of instrument-specific factors can be performed as part of a system suitability test (SST).

[0042] To account for instrument-specific factors, the NMR spectrometers used in the method of this invention must be calibrated. For this purpose, the calibration factor for each NMR spectrometer and each nuclide pair consisting of a first NMR-active nuclide and a second NMR-active nuclide is determined.

[0043] Specifically, the calibration factor can be determined as follows. First, a suitable calibration solution is prepared. This solution contains known amounts of a first NMR-active nuclide and a second NMR-active nuclide. For example, to determine the nuclide pair... 1 H, 2 The calibration factor for H is suitable for a solution of H2O in D2O, where the exact amounts of H2O and D2O contained therein are known.

[0044] Subsequently, by using the NMR spectrometer to be calibrated, a sample was generated from the calibration solution. 1 H NMR spectrum and 2 H NMR spectrum.

[0045] Then, by comparing the integrals of the respective peaks of the two spectra and taking into account the known amounts of H2O and D2O in the calibration solution, the calibration factor specific to this NMR spectrometer and this nuclide pair can be calculated.

[0046] The process of determining the calibration factor between D and all other active nuclides X can be further simplified by using specially synthesized compounds. These compounds contain nuclei D and X in amounts determined by the chemical structural properties of the compounds, and thus also by their molar ratio. Since the ratio is known, it is not necessary to weigh the sample. Such a reference material can be added in any amount to an analytical solution consisting of a weighed sample and a deuterated solvent. The calibration factor in each individual measurement is determined from the outset by the ratio of the absolute integrals of the D and X specific signals. Examples of suitable chemical compounds, such as isobutanol-d6, are listed below.

[0047] This calibration factor takes into account not only the instrument-specific factors of the NMR spectrometer used, but also the chemical and quantum mechanical factors of the aforementioned nuclide pairs. Because the calibration factor also considers instrument-specific properties, in the process according to the invention, the first and second NMR experiments and the determination of the calibration factor must be performed using the same NMR spectrometer.

[0048] Therefore, the present invention also discloses a method for calibrating an NMR spectrometer for performing the heteronuclear quantitative determination method of the present invention.

[0049] The NMR spectrometer can be calibrated before or after the first and second NMR experiments.

[0050] Finally, in the method of the present invention, the mass of the analyte contained in the sample solution is calculated, taking into account the signals of the first and second spectra and a calibration factor. This will be explained in detail below.

[0051] In the method of the present invention, preferably, the NMR active nuclide is 2 H (deuterium isotope of hydrogen), the active nuclide is a component of the deuterated solvent and is referred to above as the first NMR active nuclide.

[0052] This utilizes the fact of deuterium atoms. 2 The nuclear spin of H (or D) is 1, and therefore it can be detected by NMR spectroscopy. Therefore, any deuterated solvent can be used as an internal standard. It should be noted that... 2 The resonant frequency of H is significantly lower than that of the frequency ratio of 15.40%.1 H (relative to the proton frequency of TMS (tetramethylsilane) in CDCl3 at 0 ppm).

[0053] As explained above, 2 H has relatively low sensitivity, and 2 The sensitivity of H NMR signal measurement relative to 1 Only about 1% (9.65 × 10⁻⁶) of the H NMR signal was measured. -3 However, this is compensated for by the fact that the solvent is present in a significant excess in the sample solution during NMR spectroscopy.

[0054] If the first and second NMR experiments are performed in a pulsed manner in the method of the present invention, then when comparing signals from the two spectra, the number of pulses in the first and second NMR experiments must be taken into account (by dividing the integral value by the number of pulses).

[0055] The greatest advantage of the method of this invention is that the solvent is used as a reference standard or reference material and also as an internal standard. This eliminates the problem of NMR tube volume variation mentioned at the beginning. Since the solvent is already frequently used in NMR spectroscopy, there is no need to add another substance as a standard. The solvent, which is also used as a standard herein, only needs to be measured accurately (e.g., by measuring an accurate volume or weighing an accurate mass). This internal standard, i.e., the solvent, can also serve as a locking substance to improve frequency stability. Preferred substances herein include, for example, D₂O. Therefore, the internal standard serves simultaneously as a solvent and also as a locking substance.

[0056] Furthermore, a single pulse is usually sufficient to calibrate the measurement, i.e., record. 2 The H NMR spectrum means that the total measurement time for the entire measurement process is only delayed by about 10 seconds.

[0057] The following sections explain in detail how NMR spectral analysis of the test sample can be used to make quantitative statements about the compound, i.e., the analyte contained in the test sample, for known homonuclear quantification and heteronuclear quantification according to the invention. The variables and constants used are listed in Table 1.

[0058] Table 1

[0059]

[0060] *) The number of NMR-active nuclides that cause peaks used for quantitative determination in NMR spectra.

[0061] Calculation of isonuclear quantitative determination:

[0062] To prepare a suitable sample solution, a known mass of test sample m is weighed. PGand an internal standard of known mass m IS And it dissolves in a suitable solvent. For example, the internal standard may be a liquid, a solid, or a solution in a suitable solvent, and in a precisely known proportion (mass fraction w). RS It contains the reference material.

[0063] The structure of the reference material is known. The structure of the analyte in the test sample is also usually known or can be determined from the NMR spectrum. Therefore, individual signals / peaks visible in the obtained NMR spectrum can be clearly attributed to the reference material and the analyte, respectively.

[0064] For both the reference material and the analyte, and for each selected peak in each case, determine the integral I corresponding to the area under each peak. RS I A Furthermore, the number of atomic nuclei causing each peak must be known. For example, if dimethyl sulfone is used as a reference, its peak is located at... 1 In the 1H NMR spectrum, the peak is caused by 6 H atoms. If the analyte is quantitatively determined using the peak of a single CH3 group, the integral of this peak is caused by 3 H atoms.

[0065] The following formula (1) can be used to determine the weighing mass m of the internal standard. IS The mass fraction w of the reference substance in the internal standard RS and its molecular weight M RS To calculate the amount n of the reference substance in the sample solution. RS :

[0066]

[0067] Using the following formula (2), the integral I of a peak of the analyte and the reference material in the internal standard can be compared. A and I RS And taking into account the number of atoms ZA that respectively cause the peak A and ZA RS To calculate the amount of analyte n in the sample solution. A :

[0068]

[0069] The amount of analyte n in the sample solution can be calculated using the following formula (3). A Its molar mass M A and the weighing mass m of the test sample PG Calculate the mass fraction w of the analyte in the test sample. A :

[0070]

[0071] Calculation of heteronuclear quantitative determination:

[0072] To prepare a suitable sample solution, a known mass of test sample m is weighed. PG and a known mass of deuterated solvent m IS And it dissolves in it. Since the deuterated solvent is used as an internal standard, there is no need to add other substances to the sample solution.

[0073] Preferably, the data is recorded from the sample solution. 2 H NMR spectroscopy (using an appropriate pulse number NS) RS ) and additional NMR spectra recorded. These additional NMR spectra are typically suitable for determining the analyte (using an appropriate number of pulses N / S). A NMR spectra of ) such as 1 H or 13 C NMR spectroscopy.

[0074] Naturally, the structure of the deuterated solvent, i.e., the reference material, is known. The structure of the analyte is also usually known or can be determined from NMR spectra. Therefore, individual signals / peaks visible in the obtained NMR spectra can be clearly attributed to the reference material and the analyte, respectively.

[0075] For reference material and for 2 Integral I of selected peaks in H NMR spectrum RS Similarly, the integral I of selected peaks in other NMR spectra is measured. A In isonuclear quantitative determinations, it is necessary to know the number of atomic nuclei that cause each peak.

[0076] The following formula (1) can be used to determine the mass m of the internal standard, i.e., the deuterated solvent. IS The mass fraction w of the reference substance in the internal standard RS and its molecular weight M RS To calculate the amount n of the reference substance in the sample solution. RS :

[0077]

[0078] The mass fraction w of the reference substance RS The purity and degree of deuteration of the deuterated solvent are known, and commercially available deuterated solvents are also known.

[0079] Using the following formula (2a), the integral I of a peak of the analyte and the reference material in the internal standard can be compared. A and I RS And taking into account the number of atoms ZA that cause the peaks respectively A and ZA RS To calculate the amount of analyte n in the sample solution.A :

[0080]

[0081] Since the peak area (integral) in the NMR spectrum increases with the number of pulses, and a linear correlation can be assumed, the integral I in equation (2a) above... A and I RS Set to be respectively related to pulse number NS A and NS RS related.

[0082] Furthermore, equation (2a) takes into account factors (X, Y) that can be determined by calibration measurements. In this paper, X and Y represent the respective nuclei in the sample solution detected in the two NMR spectra, for example... 2 H and 1 H.

[0083] The amount of analyte n in the sample solution can be calculated using the following formula (3). A Its molar mass M A and the weighing mass m of the test sample PG Calculate the mass fraction w of the analyte in the test sample. A :

[0084]

[0085] In the method of this invention, the same NMR spectrometer must be used for both the first and second NMR experiments and for the determination of the calibration factor. Preferably, all other parameters that affect the integration of the peak in each NMR experiment remain constant during these measurements. Alternatively, some changes in these parameters can be mathematically accounted for and corrected for.

[0086] The parameter used to optimize the conversion of analog signals to digital signals is called RG (receiver gain). Preferably, this parameter is set to 1 for NMR experiments. In principle, the integral used in the equation is a linear function of RG, so mathematical corrections for different RG are feasible. The correction of RG must take into account the above equation 2a.

[0087] Another relevant parameter in NMR experiments is the pulse angle PW. In a fully relaxed state, the sum of the Z magnetizations from the nuclear spins is normalized to 1. The X and Y magnetizations are functions of the excitation duration, which is typically in the range of 10 to 200 microseconds. Thus, the vector Z can be induced into a circular motion. Depending on the excitation duration, this vector is also referred to as the pulse angle. With a 90° pulse, the maximum intensity excitation is achieved, and 100% of the Z magnetization is converted into X and Y magnetizations. Doubling the excitation time results in negative Z magnetization, and X and Y magnetizations are 0. In this case, the macroscopic magnetization of the excited nuclear spins describes a unit circle. In the vector field, the intensities of X and Y are sinusoidal functions. For mathematically obvious reasons, any errors that may occur due to inaccurate pulse angle setting or pulse timing are minimized with a 90° pulse. Therefore, a 90° pulse is preferred for heteronuclear quantification according to the invention. Otherwise, the absolute integral must be corrected. In the case of the same nuclear quantity, the PW has no effect because it is mathematically subtracted from the equation.

[0088] Another parameter is the so-called relaxation delay D1. It defines the experimental waiting time between two pulses (NS>1), and therefore only the case of NS>1 must be considered. This value must be large enough to ensure complete relaxation of the two measured nuclides. The relaxation time is a material property and is therefore individual for all nuclides, belonging to the same nucleus group in different molecular environments. This parameter must be observed in every quantitative assay and cannot be considered mathematically. In addition to the classic T1 measurement, there is a simple test that uses a single 90° pulse without D1 after the NMR experiment, which can be used to check whether the relaxation delay D1 is large enough.

[0089] The method of this invention elevates NMR spectroscopy to the level of a primary quantitative measurement method and is therefore equivalent to weighing. Similar to a balance, NMR experiments only require calibration.

[0090] For example, in the field of ionic active pharmaceutical ingredient analysis, the anions and cations to be analyzed can be determined qualitatively and quantitatively simultaneously through a single NMR spectroscopy analysis.

[0091] The principle of using deuterated solutions as internal standards is also the focus of this invention, and it allows for the rapid and simple determination of the degree of deuteration of deuterated compounds.

[0092] The degree of deuteration of a compound is usually given as a percentage (%), representing the proportion of hydrogen atoms in the compound that are replaced by deuterium atoms. Similarly, the degree of protonation can be determined, where: Degree of deuteration = 1 - Degree of protonation.

[0093] According to the present invention, a method for determining the degree of deuteration of a first deuterated compound can also be provided, the method comprising the steps of:

[0094] A mixture of a first deuterated compound and a second deuterated compound having a known degree of deuteration was prepared such that both deuterated compounds were dissolved.

[0095] Generate from the mixture 1 H NMR spectra were used to determine the integral of at least one peak of each of the two deuterated compounds;

[0096] Generate from the mixture 2 H NMR spectra and the integral of the same peak was measured;

[0097] The degree of deuteration of the first deuterated compound is calculated using the measured integral value and the known degree of deuteration of the second deuterated compound.

[0098] According to a preferred embodiment, at least one of the first deuteration compound and the second deuteration compound is a deuteration solvent for NMR spectroscopy.

[0099] The mixing ratio of the first and second deuterated compounds does not need to be known precisely, as long as meaningful NMR spectra are obtained. Preferably, the mixing ratio by weight is between 1:99 and 99:1, more preferably between 10:90 and 90:10, and particularly preferably between 30:70 and 70:30. The simplest and therefore particularly preferred method is to mix the first and second deuterated compounds at approximately 1:1.

[0100] From the two spectra, the integrals of the same peaks of the first and second compounds are determined, and the integral ratio D / H is calculated for each of the two compounds. Using the calculated integral ratio and taking into account the known protonation degree of the second deuterated compound, the protonation degree of the first deuterated compound and thus the degree of deuteration can be calculated by the method of equation (4).

[0101] {D} Vb1 =100-({H}) Vb2 ×(IntH Vb2 / IntD Vb2 ) / (IntH Vb1 / IntD Vb1 (4)

[0102] in:

[0103] {D} Vb1 = Degree of deuteration of the first deuterated compound

[0104] {H} Vb2 = Degree of deuteration of the second deuterated compound

[0105] IntH Vb1 = 1 Integral of the first deuterated compound peak in the 1H NMR spectrum

[0106] IntD Vb1 = 2 Integral of the same peak of the first deuterated compound in the H NMR spectrum

[0107] IntH Vb2 = 1 Integral of the second deuterated compound peak in the 1H NMR spectrum

[0108] IntD Vb2 = 2 Integral of the same peak of the second deuterated compound in the H NMR spectrum

[0109] The advantage of using this method to determine deuteration is that it does not require knowledge of the precise mixing ratio, nor of the aforementioned quantum mechanical, chemical, or instrument-specific factors. This method is particularly useful for the simple and accurate determination of deuteration in solvents used in NMR spectroscopy.

[0110] As described above, in the method for quantitative determination of heteronuclear substances using NMR spectroscopy according to the present invention, a one-time calibration of the NMR spectrometer is necessary. The calibration solution required for this purpose contains known amounts of a first NMR-active nuclide and a second NMR-active nuclide, and can therefore be prepared by accurately measuring two suitable compounds.

[0111] Alternatively, compounds that can be used for calibration may contain a defined number of two nuclides in their structural formula. For example, in 1 H and 2 When H is one of two nuclides, a suitable compound is isopropanol-d6, (CD3)2CHOH, which can be obtained by reducing acetone-d6. This is because this compound has a definite and constant set of nuclides. 2 H and 1 The ratio of H is 6:2, therefore, precise measurements are not required when using this compound as a reference.

[0112] Based on this, it can be envisioned that, in addition to a certain number of deuterium atoms, it also contains one or more other nuclides that can be detected by NMR spectroscopy, such as other chemical compounds of carbon, nitrogen, phosphorus, fluorine, silicon, boron, selenium, etc.

[0113] An example of the compound that can be used as a reference material in calibration is the phosphate ester of isopropanol-d6:

[0114]

[0115] The phosphorus mentioned therein may be other oxidation states (e.g., phosphonates).

[0116] The residue R can be selected in such a way that other NMR active nuclides, particularly fluorine, silicon, and boron, can be included as part of the reference material. In the case of R = isopropyl-d6, the resulting compound is only suitable for use as a calibration reference material in the determination of deuterium, hydrogen, and phosphorus. For the following compounds, the selection of the group R ensures that the reference material also contains a specific number of fluorine atoms, thus providing a equally suitable reference material for calibration when determining fluorine-containing compounds:

[0117]

[0118] Based on the principle of the method for quantitative determination of heteronuclear atoms using NMR spectroscopy according to the present invention, compounds as described above, i.e., compounds containing a certain number of deuterium atoms and a certain number of at least one other nuclide detectable by NMR spectroscopy in their structural formulas, are further provided for calibrating an NMR spectrometer for quantitative determination of heteronuclear atoms.

[0119] As explained above, this application also discloses a method for calibrating an NMR spectrometer used to carry out the heteronuclear quantitative determination method of the present invention. Therefore, the use of the compounds described above in such calibration methods is similarly disclosed.

[0120] Particularly preferably, the chemical compound used as a reference substance is a liquid at room temperature or slightly elevated temperatures. This makes the compound easier to use as a pure substance.

[0121] Currently, quantitative heteronuclear NMR spectroscopy involves only a purely mathematical foundation, which can be traced back to the defined natural stoichiometry of organic molecules. This system can be described as a quantum balance, and thus is a predominantly absolute analytical method, which in practical applications becomes a predominantly relative method.

[0122] The method of this invention makes it possible to perform quantitative NMR spectroscopy without adding the specific standard substances that have been used until now.

[0123] One important area of ​​this "standardless analysis" is diagnostics or forensic medicine. For example, using the solvent D₂O as a reference can significantly improve the determination of blood alcohol compared to traditional methods. For information on the determination of blood alcohol concentration using NMR spectroscopy, see patent DE 10 2012 224334B4.

[0124] Similar to the above determination of solvent deuteration, the combination... 1 H / 2 The 1H NMR method allows for the direct determination of water content in blood and plasma samples. Therefore, it eliminates the need for commonly used standards, directly determining the ethanol content (as a blood alcohol) in blood samples, and requiring only a single assay.

[0125] In addition to the quantitative determination of water and blood alcohol in blood, other important parameters of the blood composition become directly available, i.e., can be quantitatively determined in a simple and accurate manner using the method of this invention. These include glucose, lactate, specific amino acids, or ADP / ATP. The signal of complex lipoproteins is a clear marker of human blood, which can also be detected and form a spectral fingerprint, which, especially in forensic samples, identifies the identity and quantity of the examination sample as human blood. Similarly, using appropriate measurement conditions, other metabolites, including gamma-hydroxybutyric acid (GHB), also known as liquid ecstasy, can be detected. Attached Figure Description

[0126] Other advantages and features of the present invention can be seen from the description of the embodiments and from the figures, wherein:

[0127] Figure 1 This shows the Na-heparin in D2O 2 H NMR spectroscopy.

[0128] Figure 2 This shows the Na-heparin in D2O 23 Na NMR spectroscopy.

[0129] Figure 3 Different concentrations of sodium heparin were shown. 23 Na NMR spectroscopy.

[0130] Figure 4 A magnified view of DMSO-d6 with detailed images is shown. 1 H NMR spectroscopy.

[0131] Figure 5 The DMSO-d6 is shown 2 H NMR spectrum (left) and magnified corresponding Figure 4 of 1 Overlap of H NMR spectra (right).

[0132] Figure 6 A graph showing the degree of deuteration of deuterated DMSO-d6 is presented.

[0133] Figure 7 A mixture of DMSO-d6 and CDCl3 is shown. 1 H NMR spectrum (left) and corresponding 2 H NMR spectrum (right). Detailed Implementation

[0134] Example 1 - Determination of Sodium in Heparin Sodium

[0135] The determination of sodium in heparin sodium is provided below as an example of the method of the present invention, namely, heteronuclear quantitative determination.

[0136] For this purpose, the sodium content of 16 different sodium-heparin solution samples (S1-S16) was determined by the method (NMR) according to the present invention and compared with the values ​​obtained by atomic absorption spectrometry (AAS), which is commonly used for this purpose.

[0137] To prepare the sample, a measured amount of Na-heparin is weighed and dissolved in a measured volume of D₂O. Alternatively, a measured amount of a suitable deuterating solvent can be weighed. DMSO-d₆ can be used as a substitute for D₂O as a deuterating solvent.

[0138] In particular, the determination of sodium by NMR according to this invention is required by the European and United States Pharmacopoes. 1 Additional experiments were conducted following the H NMR studies. For this purpose, existing sample solutions could be used without additional sample preparation, thus requiring only about 2 minutes of additional time per measurement.

[0139] NMR experiments produce 2 The time required for H NMR spectroscopy is less than 10 seconds. An example of Na-heparin in D2O. 2 H NMR spectrum is shown in Figure 1 It is generated in a single pulse using a Prodigy-type 500MHz spectrometer (manufactured by Bruker Corporation). The signal-to-noise ratio (S / N) is 17000.

[0140] NMR experiments produce 23 The time required for Na NMR spectroscopy is approximately 2 minutes. An example of Na-heparin in D2O. 23 Na NMR spectrum is shown in Figure 2 It was generated in 128 pulses using a Prodigy-type 500MHz spectrometer (manufactured by Bruker Corporation). The signal-to-noise ratio (S / N) is 4900.

[0141] exist Figure 3 shown 23 In Na NMR spectroscopy, Na signals from different samples with different Na heparin concentrations are superimposed.

[0142] Based on each 2 H NMR and 23 The signal in the Na NMR spectrum (integration), taking into account the NMR spectrometer and nuclide pair used. 2 H, 23 The calibration factor for Na was used to determine the Na content in each sample.

[0143] Table 1 below shows a comparison between the results obtained by the method of the present invention and the results determined by atomic absorption spectrometry (AAS).

[0144] Table 1

[0145]

[0146] It was found that, within the range of measurement accuracy, the results of NMR detection were consistent with those of the classical AAS method used for Na.

[0147] The process described above can be easily converted to all other active NMR nuclides, for example 35 Cl or 79 Br.

[0148] Example 2 - Determination of NMR solvent deuteration

[0149] The appropriate solvent for NMR experiments must contain a very high level of deuterium. 2 H, rather than the more naturally abundant hydrogen atoms 1 H. For traditional NMR experiments, "very high" specification is sufficient, i.e., >99%. D. The remaining 1% is... 1 H.

[0150] Understanding this degree of deuteration is important for the heteronuclear quantitative NMR method according to the present invention. In cases where the solvent has a very high degree of deuteration, for example, D₂O with a deuteration degree >99.95%, 0.05%... 1 The proportion of H is unimportant; it only affects the uncertainty of the measurement. For accurate determination of deuteration, NMR spectroscopy is naturally suitable because it can be used to measure... 2 H(D) and 1 Both H and H. The integral ratio D / H is directly proportional to the degree of deuteration. Necessary calibration can be performed by adding the same amount of non-deuterated solvent and performing appropriate calculations.

[0151] The following explanation uses deuterated DMSO-d6 (CD3-SO-CD3) as an example. According to the manufacturer, the degree of deuteration of the DMSOd6 used in this example is approximately 99.5%. Therefore, some DMSO molecules do not contain two identical CD3 groups, but also contain CD2H or CDH2 groups. 1 In the 1H NMR spectrum, the signal of the CD2H group (due to...) 2 J D,H The quintet generated by coupling) and the CDH2 signal (due to 2 J D,H The triplet generated by coupling can be displayed separately (see...) Figure 4 ).exist 2In the H NMR spectrum, only the major CD3 group is visible (see...). Figure 5 ).

[0152] Figure 4 In the middle, DMSO-d6 1 The H NMR spectrum is shown on the left. Figure 4 In the middle, a vertical magnification reveals a triplet peak for the CHD2 group. After adding undeuterated DMSO to this sample, the singlet peak for the CH3 group shifted to the deeper field. This is attributed to the so-called deuterium isotope effect. 1 The equidistant shift of the H resonance results in the same displacement in each deuterium direction of the high field.

[0153] Because the CH3 groups in sulfur also show a separated signal (due to the loss of...) 2 J D,H The resulting singlet, under the current circumstances, is best addressed by using conventional DMSO as both an internal standard and a reference standard, which is suitable for accurately determining the proton content in DMSO-d6. Knowing the amount of residual protons, which can be determined very precisely using this method, allows for easy calibration of the deuterium / hydrogen ratio. Therefore, the deuterated solvent used to accurately determine the degree of deuteration in this method can be used as a standard for heteronuclear quantification in the method of this invention.

[0154] Figure 5 DMSO-d6 is shown on the left. 2 H NMR spectra, and on the right are shown the comparison with those from... Figure 4 The corresponding 1 Magnified and superimposed HNMR spectra. The spectrum on the right is magnified vertically to show... 13 C NMR satellite peak.

[0155] After different amounts of undeuterated DMSO were added to the deuterated DMSO-d6 used in this embodiment, each of the resulting mixtures was measured by NMR spectroscopy and the integral ratio D / H was determined. The D / H ratio obtained thereafter was plotted relative to the proportion of the added undeuterated DMSO (in weight %). The degree of deuteration of the DMSO-d6 used could be calculated by a linear correction equation of the following equation (5).

[0156] Deuteration degree = 100 – intercept / slope (5).

[0157] Figure 6 A graph showing the measurements obtained according to this embodiment and the corresponding line created using linear regression is shown. The coefficient of determination R... 2 The value is 0.9999. Using the linear equation y = 2.7877x + 1.194, the degree of deuteration of the deuterated DMSO-d6 used is 99.57%.

[0158] Now, the solvents that are precisely specified in terms of deuteration degree as described in Example 2 above can be used as general standards for determining other deuterated solvents such as CDCl3, and thus other standards for the method of the present invention (heteronuclear quantification) can be specified.

[0159] For this purpose, a solvent with a specified degree of deuteration is mixed with the previously defined DMSO-d6, and the appropriate steps are performed. 1 H and 2 H NMR spectroscopy measurements. The mixing ratio does not need to be known because all parameters affecting the measurement (e.g., quantity, individual instrument settings, etc.) have been taken into account. This process is explained in detail in Example 3 below.

[0160] Example 3 - Determine the deuteration degree of the NMR solvent using other deuterated solvents with known deuteration degrees.

[0161] In Example 3, the degree of deuteration of deuterated CDCl3 was determined. For this purpose, a mixture of deuterated CDCl3 and DMSO-d6 (99.57% deuteration) used in Example 2 was prepared at a mixing ratio of approximately 1:1. Since precise knowledge of the mixing ratio is not required, the mixing ratio can be weight-dependent or volume-dependent. Other mixing ratios can also be used, as long as meaningful NMR spectra can be obtained. The measured NMR of the resulting mixture... 2 H and 1 H NMR (one single pulse for each).

[0162] Figure 7 The prepared mixture of CDCl3 and DMSO-d6 is shown on the left. 1 H NMR spectra, and the corresponding H NMR spectra are shown on the right. 2 H NMR spectrum.

[0163] The integral ratio D / H from the DMSO-d6 spectrum was compared with the integral ratio D / H from the CDCl3 spectrum, taking into account the known degree of protonation ({H} of DMSO-d6). DMSO The protonation and deuteration degrees of CDCl3 were calculated. Table 2 shows the corresponding measurements and calculation results.

[0164] Table 2

[0165]

[0166] Degree of deuteration of chloroform {D} CDCl3 The percentage can be calculated using the following equation (6):

[0167] {D} CDCl3 = 100 - ({H} DMSO × (IntH DMSO / IntDDMSO ) / (IntH CDCl3 / IntD CDCl3 (6).

[0168] Therefore, the degree of deuteration of the deuterated chloroform was obtained to be 99.83%.

Claims

1. A method for determining the degree of deuteration of a first deuterated compound, the method comprising the following steps: A mixture of a first deuterated compound with an unknown mixing ratio and a second deuterated compound with a known degree of deuteration was prepared, such that both deuterated compounds were dissolved. Generate from the mixture 1 H NMR spectra were used to determine the integral of at least one peak of each of the two deuterated compounds; Generate from the mixture 2 H NMR spectra and the integral of the same peak was measured; The degree of deuteration of the first deuterated compound is calculated using the measured integral value and the known degree of deuteration of the second deuterated compound. The degree of deuteration of the first deuterated compound is calculated using equation (4): {D} Vb1 =100-({H} Vb2 ×(IntH Vb2 / IntD Vb2 ) / (IntH Vb1 / IntD Vb1 (4) in: {D} Vb1 = Degree of deuteration of the first deuterated compound {H} Vb2 = Degree of deuteration of the second deuterated compound IntH Vb1 = 1 Integral of the first deuterated compound peak in the 1H NMR spectrum IntD Vb1 = 2 Integral of the same peak of the first deuterated compound in the H NMR spectrum IntH Vb2 = 1 Integral of the second deuterated compound peak in the 1H NMR spectrum IntD Vb2 = 2 The integral of the same peak of the second deuterated compound in the H NMR spectrum.

2. The method according to claim 1, wherein at least one of the first deuterated compound and the second deuterated compound is a deuterating solvent for NMR spectroscopy.

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